The present invention relates to methods and systems for delivering a nucleic acid molecule into a target cell, and more particularly to methods and systems for delivery with spatial or temporal control, or both.
While the nucleic acid delivery methods and systems of the invention apply generally to target cells capable of taking up nucleic acid from outside the cells, spatial and temporal control offers particular utility when applied in the context of cell differentiation, especially in bioengineering three-dimensional matrices such as tissues or organs containing multiple differentiated cell types that derive from a single stem cell precursor. Early strategies for directed cell differentiation focused on supplementing culture environments with growth factors, signaling molecules and extracellular matrix components. The interplay among target cells (especially stem cells), growth factors, signaling molecules and extracellular matrix components has shed light on the factors and conditions required to produce differentiated cells in sufficient numbers for regenerating a tissue of interest. More recent nucleic acid delivery strategies involve introducing nucleic acid molecules that encode the required factors, molecules and/or extracellular matrix components into cells.
Recent publications have reported limited spatial and/or temporal control over nucleic acid delivery to a desired cell population in culture. For example, in U.S. Pat. No. 6,890,556 (Segura et al.) and in published US Patent Application No. 2005/0090008 (Segura et al.), each incorporated herein by reference as if set forth in its entirety, polyanionic residues of a nucleic acid molecule (DNA, RNA or oligonucleotide) interact strongly with a polycationic polylinker (polymer, protein, peptide or lipid) to form a nucleic acid-polylinker non-covalent complex. The complex is covalently or non-covalently tethered to a support substrate that also supports cell adhesion.
The system described in the above-noted documents provides some spatial and temporal control over nucleic acid delivery by controlling both the strength of the interactions between the nucleic acid and the polylinker, as well as the location of the complexes relative to the adhered cells. For example, one can control the density of the polylinker or of the tether on the substrate, or one can select a polylinker or a tethering agent having stronger or weaker binding characteristics, as described therein.
Several investigators have shown that co-localization of cells and plasmid DNA—via immobilization of plasmid DNA to cell culture substrates—substantially enhances gene uptake and transgene expression, both in vitro and in vivo. See Shen H, et al., “Surface-mediated gene transfer from nanocomposites of controlled texture,” Nat. Mater. 3:569-574 (2004); Segura & Shea, supra; Segura T, et al., “Substrate-mediated DNA delivery: role of the cationic polymer structure and extent of modification,” J. Control Release 93:69-84 (2003); Bengali Z, et al., “Gene delivery through cell culture substrate adsorbed DNA complexes,” Biotechnol. Bioeng. 90:290-302 (2005); Chang F, et al., “Surfection: a new platform for transfected cell arrays,” Nucleic Acids Res. 32:e33 (2004); Delehanty J, et al., “A comparison of microscope slide substrates for use in transfected cell microarrays” Biosens. Bioelectron. 20:773-779 (2004); Delehanty J, et al., “Transfected cell microarrays for the expression of membrane-displayed single-chain antibodies,” Anal. Chem. 76:7323-7328 (2004); Kato K, et al., “Transfection microarray of nonadherent cells on an oleyl poly(ethylene glycol) ether-modified glass slide,” Biotechniques 37:444-448, 450, 452 (2004); Bielinska A, et al., “Application of membrane-based dendrimer/DNA complexes for solid phase transfection in vitro and in vivo,” Biomaterials 21:877-887 (2000); and Ziauddin J & Sabatini D, “Microarrays of cells expressing defined cDNAs,” Nature 411:107-110 (2001). However, previous immobilization approaches typically use non-specific interactions that complicate immobilization of multiple distinct plasmids. Additionally, the affinity of the plasmid DNA for the substrate is typically very high (e.g., pM dissociation constants for avidin-biotin-based DNA immobilization) and not readily controllable. Because the existing systems use non-specific polyanionic-polycationic interactions to hold the nucleic acid in place, the systems offer no mechanism for sequence-specific nucleic acid patterning on the supporting substrate.
It is advantageous to exert greater and more flexible spatial and temporal control over inductive growth factor production, and to enable production of multiple growth factors in a controlled manner in a material that can appropriately support new tissue growth and development.